In order to decrease morbidity after surgery, it is critical that pain treatment strategies that have greater efficacy and are devoid of serious side effects be developed for postoperative patients. The long-term goal of this proposal is to understand how factors that activate and sensitize pain transmitting neurons can be effectively targeted for the improved treatment of pain in patients undergoing surgery. The overall objective of this proposal is to demonstrate powerful activation of nociceptive pathways by deep muscle incision and sensitization of nociceptive pathways to ischemic pain mediators and to muscle contraction by incision. The central hypothesis is that incisions that include deep muscle tissue produce much greater activation of the nociceptive system than incisions of skin only. The rationale that underlies the proposed research is that it will be possible to understand the mechanisms and origin of pain at rest and pain with activities in patients after surgery. To accomplish the overall objective, the following specific aims are proposed: 1. Demonstrate unique pain-related behaviors are produced by deep muscle incision. The working hypothesis, supported by preliminary studies, is that deep muscle incision will cause guarding pain and will reduce activities requiring muscle contraction. Skin incision will not affect these behaviors. 2. Demonstrate activation and sensitization of primary afferent nociceptors by deep muscle incision but not by skin incision. Based on previous studies, skin incision does not produce sustained activation of nociceptors. We hypothesize that nociceptors from the hindpaw of rats with deep muscle incision will have ongoing activity and will be sensitized to muscle contractions. 3. Demonstrate greater excitability of incised muscle compared to unincised muscle in a rat hindpaw in vitro muscle- nerve preparation. We hypothesize deep tissue incision will sensitize nociceptors and this will be evident in vitro. We will record nociceptors in vitro and examine excitation of nociceptors by heat, mechanical stimuli, and acid as well as muscle contraction. The effect of ASIC and TRPV1 receptor blockade on excitation by acid will be tested. 4. Demonstrate DRG innervating incised muscle have increased responses to acid and increased mRNA expression for ASICs and TRPV1 compared to DRG innervating unincised muscle. We hypothesize that 1) the percentage of DRG expressing ASIC and TRPV1 currents and/or the amplitude of the ASIC- and TRPV1-like currents will be greater in incised muscle, and 2) mRNA expression for ASICs and TRPV1 will be greater in incised muscle. 5. Demonstrate greater excitability of incised muscle compared to unincised muscle in a mouse in vitro muscle nerve preparation. We hypothesize murine muscle nociceptors will be sensitized by incision. This aim will provide the basis for future studies that will incorporate KO mice for key receptors and mediators of nociceptor activation and sensitization by muscle incision. It is expected that strong activation and sensitization of nociceptive pathways by incision of deep muscle tissues will be demonstrated. The proposed research is significant because it will demonstrate that after incision, sensitization of nociceptors to chemical stimuli and muscle contractions occurs. From this research, ischemic pain mechanisms and therapeutic targets like TRPV1 and ASICs can be advanced. PUBLIC HEALTH RELEVANCE: Pain from surgery on muscle tissue will be studied. From this research, new mechanisms and treatments will become available so that pain at rest and with movement will be reduced, perioperative morbidity will decline, and health care costs will decrease.